The objectives of this research program is to contribute to an understanding of the molecular basis of the biological activities of membrane bound proteins. These studies will focus on the enzyme system involved in the degradation of proteins in the membrane, and the enzyme complex responsible for glycosylation of the proteins transported out of the membrane or that are oriented towards the lumen of the membrane. Continue studies of electron transport enzymes of endoplasmic reticulum, including the drug metabolizing enzymes, the flavin-containing monooxygenases (FMO). Studies will be extended to the newly recognized dehydrogenases and reductases of the lumen of endoplasmic reticulum. It is intended to achieve a detailed description of the structures of both active, catalytic domains and segments that participate in interactions with the lipid bilayer, and their protein components to yield an active complex. These objectives will require not only application of protein chemistry, but also increased use of a variety of physical, chemical, immunological and molecular biology methods to achieve a relatively complete definition of the active sites, protein-protein interactions, and functional orientation. The spectrum of methodologies will include various mass spectrometry techniques, bifunctional cross linking, expression vectors for these enzymes and site-directed mutagenesis. In addition to the basic information concerning membrane structure that these studies should generate, the system has more direct implications for health related problems, since altered, but functionally active gene products, such as the cystic fibrosis transmembrane conductance regulator protein are not transported to their cellular positions, but are retained in the endoplasmic reticulum and rapidly degraded. The signals for the retention and the enzymes responsible for the degradation are unknown. Many proteins as well as viral proteins pass through the membrane to reach their sites of assembly. Many undergo proteolysis, glucosylation, acylation and deacylation. Thus, the basic studies addressed in this proposal should provide fundamental knowledge relevant to the clinical problems of premature proteolysis of altered gene products, atherosclerosis (steroyl CoA desaturase, and drug metabolism (esterases and FMO's)). Elucidation of the proteolysis and glycosylation processes of the endoplasmic reticulum may also provide investigators to design targets to accelerate or to inhibit glycosylation and proteolytic destruction of specific intrinsic or foreign viral proteins.
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